Lyophilized polyethylene oxide modified catalase composition, polypeptide complexes with cyclodextrin and treatment of diseases with the catalase compositions

ABSTRACT

Disclosed are lyophilized biologically active proteinaceous compositions containing low diol polyalkylene oxide, such as polyethylene glycol, covalently attached to a biologically active proteinaceous substance and combined with the cryoprotectant cyclodextrin.

This application is a continuation-in-part of application Ser. No.08/178,205, filed on Jan. 5, 1994 which, in turn, is a division ofapplication Ser. No. 08/023,182 filed on Feb. 25, 1993 now U.S. Pat. No.5,298,410.

BACKGROUND OF THE INVENTION Field of the Invention

This invention relates to lyophilized aqueous parenteral solutions ofphysiologically active proteins and polypeptides attached to low diolpolyalkylene oxide combined with the cryoprotectant cyclodextrin.

More particularly, this invention relates to a lyophilized aqueousparenteral solution of superoxide dismutase attached to low diolpolyethylene glycol combined with the cryoprotectant cyclodextrin, andto a lyophilized aqueous parenteral solution of catalase attached to lowdiol polyethylene glycol combined with the cryoprotectant cyclodextrin.

Reported Developments

Biologically active proteins, particularly enzymes and peptide hormones,have been long considered as ideal drugs for the treatment of variousdiseases due to their specificity and rapid catalytic action. Suchenzymes include:

Oxidoreductases such as: Urate: oxygen oxidoreductase (1.7.3.3;"uricase"); Hydrogen-peroxide: hydrogen-peroxide oxidoreductase(1.11.1.6; "catalase"); Cholesterol, reduced-NADP: oxygen oxidoreductase(20-β-hydroxylating) (1.14.1.9; "Cholesterol 20-hydroxylase").

Transferases such as: UDP glucuronate glucuronyl-transferase (acceptorunspecific) (2.4.1.17; "UDP glucuronyltransferase"); UDP glucose:α-D-Galactose-1-phosphate uridylyltransferase 2.7.7.12).

Hydrolases such as: Mucopeptide N-acetylmuramyl-hydrolase (3.2.1.17;lysozyme); Trypsin (3.4.4.4); L-Asparagine aminohydrolase (3.5.1.1;"Asparaginase").

Lyases such as: Fructose-1,6-diphosphateD-glyceraldehyde-3-phosphate-lyase (4.1.2.12; "aldolase").

Isomerases such as D-Xylose ketol-isomerase (5.3.1.5; xylose isomerase)and

Ligases such as: L-Citrulline: L-aspartate ligase (AMP) (6.3.4.5).

The peptide hormones include:

Insulin, ACTH, Glucagon, Somatostatin, Somatotropin, Thymosin,Parathyroid hormone, Pigmentary hormones, Somatomedin, Erythropoietin,Luteinizing hormone, Chorionic Gonadotropin, Hypothalmic releasingfactors, Antidiuretic hormones, Thyroid stimulating hormone, Calcitoninand Prolactin.

Therapy with physiologically active proteinaceous substances,particularly with non-human enzymes, has been less than successful duein part to their relatively short half-lives and to their respectiveimmunogenicities. Upon administration, the host defense system respondsto remove the foreign enzymes by initiating the production of antibodiesthereto, thereby substantially reducing or eliminating their therapeuticefficacies. Repeated administration of foreign and of otherwise shortlived human enzymes is essentially ineffective, and can be dangerousbecause of concomitant allergic response. Various attempts have beentaken to solve these problems, such as through microencapsulation,entrapment in liposomes, genetic engineering and attachment of theenzymes to polymers. Among the attempts the most promising appears to bethe chemical attachment of the proteinaceous substances to polyalkyleneoxide (PAO) polymers and particularly polyethylene glycols (PEG). Thefollowing illustrates these attempts.

U.S. Pat. No. 4,179,337 discloses the use of polyethylene glycol orpolypropylene glycol coupled to proteins to provide a physiologicallyactive non-immunogenic water soluble polypeptide composition in whichthe polyethylene glycol (hereinafter sometimes referred to as PEG)serves to protect the polypeptide from loss of activity without inducingsubstantial immunogenic response. The methods described in the patentfor the coupling of polyethylene glycol to a protein involve either theconversion of a protein amino group into an amide or pseudoamide, withconsequent loss of charge carrying capacity of the amino group, or theintroduction at the amino group of the protein, or vicinal to it, of aheteroatom substituent such as a hydroxyl group or of a ring system thatis not repeated in the polymer backbone.

Veronese, F. M., Boccu, E., Schaivon, O., Velo, G. P., Conforti, A.,Franco, L., and Milanino, R., in Journal of Pharmacy and Pharmacology,35, 757-758 (1983), reported that when bovine erythrocyte derivedsuperoxide dismutase is modified with a polyethylene glycol carboxylicacid N-hydroxysuccinimide active ester, the half-life of the enzyme inrats is increased over that of the unmodified protein.

European Patent Application 0 200 467 of Anjinomoto, Inc. describessuperoxide dismutase that is chemically modified by a polyalkylene oxide(PAO) which is functionalized at both ends of the polymer with activatedcarboxyl coupling groups, each capable of reacting with protein. Becausethe activated coupling sites are located at opposite ends of the polymerchain, it is unlikely that the presence of an activated group at one endof the polymer can have a significant effect on the reactive nature ofthe group at the other end of the polymer. These polymers are capable ofreacting at both ends to cross-couple with proteins to form copolymersbetween the protein and the polyalkylene oxide. Such copolymers do nothave well defined or molecularly stoichiometric compositions.

Veronese, F. M. et al in Journal of Controlled Release, 10, 145-154(1989) report that the derivatization with monomethoxypolyethyleneglycol (hereinafter sometimes referred to as MPEG) of superoxidedismutase (hereinafter sometimes referred to as SOD) gives aheterogenous mixture of products. Heterogeneity was demonstrated todepend on the presence of bifunctional polyethylene glycol (DPEG) in themonofunctional methoxylated molecules.

These attempts, in general, have resulted in somewhat longer half-lifeand reduced immunogenicity of the proteinaceous physiologically activesubstances. However, it appears that further improvements are necessaryto successfully treat a variety of diseases with these promisingbiological substances.

In co-pending patent application Ser. No. 07/936,416 (which isincorporated herein by reference) it is disclosed that biologicallyactive proteinaceous substances can be made to possess longer half-lifeand less immunogenic properties by chemically modifying them using lowdiol polyalkylene oxide, preferably polyethylene glycol. Theformulations disclosed have distinct advantages over the prior artdisclosed formulations of polyethylene glycol-modified, proteinaceoussubstances.

During storage in the liquid state, polyethylene glycol proteinaceousmolecules are hydrolyzed to a mixture of free polyethylene glycol,polyethylene glycol-protein and succinate-protein moieties. To preventsuch a destabilization process, the formulations may be lyophilized.With lyophilization, however, the concentration of protein andstabilizers is at high levels and, depending on the excipients employed,deleteriously influence the degree of intermolecular aggregation thatoccurs during storage.

It has been found that cyclodextrins inhibit the rate of intermolecularaggregation of covalently attached low diol polyethylene glycol-proteinsduring their storage, and therefore, provide for extended shelf-life.

Cyclodextrins are known in the art to possess the ability to forminclusion complexes and to have concomitant solubilizing properties.Derivatives of cyclodextrins are also known to possess these properties.Their use is illustrated by the following patents.

U.S. Pat. No. 4,596,795, relates to the administration of sex hormonesin the form of their complexes with hydrophilic derivatives ofcyclodextrin, such as poly-β-cyclodextrin andhydroxypropyl-β-cyclodextrin, by sublingual or buccal routes. Thecomplexes were found highly water-soluble and effective by comparison toother cyclodextrin derivatives.

U.S. Pat. No. 4,727,064 discloses pharmaceutical preparations consistingof a drug having low water solubility and an amorphous water-solublecyclodextrin-based mixture. The addition of the cyclodextrin-basedmixture improves the dissolution properties of the drug. Thecyclodextrin-based mixture is prepared form α-, β- or γ-cyclodextrinwhich were rendered amorphous through non-selective alkylation.

International Application No. PCT/US89/04099 (WO 90/03784) describes alyophilized composition comprising a polypeptide and astabilizing/solubilizing amount of cyclodextrin selected from the groupconsisting of hydroxypropyl, hydroxyethyl, glucosyl, maltosyl andmaltotriosyl derivatives of β- and γ-cyclodextrin.

U.S. Pat. No. 4,983,586 discloses a method for decreasing the incidenceof precipitation of a lipophilic and/or water-labile drug occurring atthe injection site, when the drug is being parenterally administered,comprising administering the drug in an aqueous solution containingabout 20% to 50% hydroxypropyl-β-cyclodextrin.

A large number of drugs are claimed including: antineoplastics,sedatives, tranquilizers, anticonvulsants, antidepressants, hypnotics,muscle relaxants, antisposmodics, anti-inflammatories, anticoagulants,cardiotonics, vasodilators and anti-arrhythmics.

We have surprisingly found that lyophilized parenteral formulationscomprising conjugated low diol polyoxyethylene oxide and aphysiologically active protein or polypeptide in a complex withcyclodextrin provide stability on extended shelf-life to theformulations without intramolecular aggregation.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides stable parenteralformulations of physiologically active proteins covalently bound to lowdiol polyalkylene oxide (hereinafter sometimes referred to as LDPAO),preferably low diol polyethylene glycol (hereinafter sometimes referredto as LDPEG) complexed with cyclodextrins.

More specifically, the present invention is directed to a pharmaceuticalcomposition comprising:

from about 150 to about 150,000 U/ml, preferably,

from about 25,000 to about 150,000 U/ml, most preferably,

from about 50,000 to about 150,000 U/ml of a covalently bound low diolpolyethylene oxide/protein;

from about 0.1 to about 20% w/v, preferably,

from about 1.0 to about 15% w/v, and most preferably,

from about 5 to about 10% w/v of cyclodextrin; and

from about 0.01 to about 50 mM of a buffer at a pH of 5.7 to 6.5.

As used herein, the term "U/ml" means enzymatic activity expressed ininternational units (U) per milliliter of a liquid composition.

As used herein, the term "low diol" with respect to a polyalkyleneoxide, such as polyethylene glycol, refers to a linear polyalkyleneoxide containing not more than about 10% of non-monoalkoxylatedpolyalkylene oxide, preferably non-monomethoxylated polyethylene glycol.

"Covalent bond" denotes the conjugate of polyalkylene oxide and thebiologically active protein.

The preferred low diol polyethylene oxide used in the present inventionis a polyethylene glycol polymer having average molecular weights offrom about 1,000 to about 15,000 daltons and containing not more thanabout 10% w/w of non-monomethoxylated polyethylene glycol are especiallysuitable for covalent attachment to biologically active proteins,especially to superoxide dismutase. More preferably, polyethyleneglycols having average molecular weights of from about 2,000 to about10,000 daltons and most preferably of from about 4,000 to about 6,000daltons are used in the present invention wherein the polyethyleneglycol preferably contains less than about 7% w/w and most preferablyless than about 5% w/w non-monomethoxylated polyethylene glycol.

The biologically/physiologically active proteins, polypeptides andhormones used in the present invention include:

Recombinant human interleukin-4 (rhuIL-4);

Protease Subtilisin Carlsberg;

Superoxide dismutases such as bovine, human, and various recombinantsuperoxide dismutases such as recombinant human superoxide dismutase(rhuSOD);

Oxidoreductases such as: Urate: oxygen oxidoreductase (1.7.3.3;"uricase"); Hydrogen-peroxide: hydrogen-peroxide oxidoreductase(1.11.1.6; "catalase"); Cholesterol, reduced-NADP: oxygen oxidoreductase(20-β-hydroxylating) ( 1.14.1.9; "Cholesterol 20-hydroxylase");

Transferases such as: UDP glucuronate glucuronyl-transferase (acceptorunspecific) (2.4.1.17; "UDP glucuronyltransferase"); UDP glucose:α-D-Galactose-1-phosphate uridylyltransferase 2.7.7.12);

Hydrolases such as: Mucopeptide N-acetylmuramyl-hydrolase (3.2.1.17;lysozyme); Trypsin (3.4.4.4); L-Asparagine aminohydrolase (3.5.1.1;"Asparaginase");

Lyases such as: Fructose-1,6-diphosphateD-glyceraldehyde-3-phosphate-lyase (4.1.2.12; "aldolase");

Isomerases such as D-Xylose ketol-isomerase (5.3.1.5; xylose isomerase);and

Ligases such as: L-Citrulline: L-aspartate ligase (AMP) (6.3.4.5).

Insulin; ACTH; Glucagon; Somatostatin; Somatotropin; Thymosin;Parathyroid Hormone; Pigmentary Hormones; Somatomedin; Erythropoietin;Luteinizing Hormone; Chorionic Gonadotropin; Hypothalmic ReleasingFactors; Antidiuretic Hormones; Thyroid Stimulating Hormone; Calcitonin;Prolactin; Interferons (alpha, beta and gamma); Antibodies (IgG, IgE,IgM, IgD); Intefieukins 1, 2, 3, 4 and 7; Granulocyte Colony StimulatingFactor (GCSF); Granulocyte-Macrophage Colony Stimulating Factor(GM-CSF); Tumor Necrosis Factor (TNF); Platelet Derived Growth Factor(PDGF); Epidermal Growth Factor (EGF); Nerve Growth Factor (NGF); BoneGrowth Factor (BGF); Growth Hormone Releasing Factor (GHRF); Papain;Chymotrypsin; Thermolysin; Streptokinase and Activase.

Cyclodextrins used in the present invention are α-, β- andγ-cyclodextrins composed of 6, 7 and 8 glucose units respectively. Theyare known in the art and recognized to possess the ability to forminclusion complexes with certain drugs, proteins and polypeptides and tohave concommitant solubilizing properties.

The inside cavity of cyclodextrin is lipophilic, while the outside ofthe cyclodextrin is hydrophilic. Because of these properties they havebeen used in forming inclusion complexes with pharmaceuticals. For thepurpose of stabilizing the low diol polyalkylene oxide/proteinconjugates hydroxyethyl, hydroxypropyl, glucosyl, maltosyl andmaltotriosyl derivatives of β-cyclodextrin are especially suitable.

The pharmaceutically acceptable aqueous carrier utilized by the presentinvention is a non-toxic, inert medium in which is dissolved the complexof cyclodextrin low diol polyethylene oxide/peptide conjugate and apharmaceutically acceptable buffer, such as sodium phosphate, sodiumacetate, sodium carbonate and those derived from mineral and organicacids. By pharmaceutically acceptable buffers it is meant that thebuffers are relatively innocuous to the mammalian organism in medicinaldoses of the buffers so that the beneficial properties of the activecomplex are not vitiated by side effects ascribable to the buffers.

DETAILED DESCRIPTION OF THE INVENTION

In the process of making the formulations of the present invention,first, the low diol polyethylene glycol is covalently attached to thebiologically active protein as shown schematically:

a) LDPEG+carboxylating agent→LDPEG--COOH

b) LDPEG--COOH+carboxyl group activating agent→active ester ofLDPEG--COOH

c) n (active esters of LDPEG--COOH)+Protein→(LDPEG--CO)_(n) -Protein

wherein:

LDPEG--COOH is LDPEG carboxylated at hydroxyl sites; and n is the numberof sites of attachment of LDPEG to protein.

LDPEG is carboxylated at the hydroxyl sites, then the carboxyl groupsare esterfied with a carboxyl activating agent to form the active esterswhich are then coupled to the protein molecule. The number of LDPEGmolecules attached to the protein will vary according to the number ofreactive groups, such as amino groups, present on the protein molecule.

The LDPEG is then dissolved in a pharmaceutically acceptable aqueouscarrier, followed by the addition and dissolution of the desiredcyclodextrin. The solution is then freeze-dried in a lyophilizer. Thelyophilized solution is reconstituted with sterile water prior to itsadministration to the patient.

The invention will be described with specific reference to superoxidedismutase (hereinafter sometimes referred to as SOD), and tohydrogen-peroxide oxidoreductase (hereinafter referred to as catalase).

Superoxide dismutase is an intracellular enzyme present in alloxygen-metabolizing cells and is responsible for catalyzing theconversion of the superoxide radical to oxygen and hydrogen peroxide.The superoxide radical and species derived from it are believed to becausative agents in a wide variety of inflammatory disorders. Superoxidedismutase is being used to treat certain inflammatory conditions underthe tradename of Orgotein. In addition, the use of SOD has beeninvestigated for broncho-pulmonary dysplasia and hyperbaric oxygentoxicity, acute inflammation caused by burns and infections, reperfusioninjury following organ transplants, retrolental fibroplasia, sideeffects of therapeutic ionization radiation and certain dermatologicalconditions. However, when SOD is administered by intravenous injectionto a mammal, the enzyme's half-life is only a few minutes and itdisappears from circulation. As a result, the enzymatic activity is notsufficient to remove toxic substances from the bloodstream. Repeatedadministration on the other hand causes adverse reactions.

Low diol polyalkylene oxide comprising chains of polyalkylene oxide ofvarying molecular weight and containing at least one hydroxyl group perchain, such as low diol polyethylene glycol (LDPEG) is attached tosuperoxide dismutase (SOD) to form a biologically active compositionhaving longer half-life and less immunogenicity than either native SODor a PAO-SOD composition. Upon lyophilization, LDPEG forms undesirableaggregates which affect its biological activity. Its complexation withcyclodextrin eliminates aggregate formation and the reconstitutedformulation is rendered stable on extended shelf-life.

The process of attaching LDPEG to SOD (sometimes hereinafter referred toas LDPEGation) comprises the steps of:

activating low diol methoxy-PEG, having an average molecular weight offrom about 1,000 to about 15,000, more preferably of from about 2,000 to10,000, and most preferably from about 4,000 to 6,000 daltons,containing not more than about 10% non-monomethoxylated PEG, bysuccinylation to form LDPEG-succinate (LDPEG-S), preferably withsuccinic anhydride (SA), followed by the formation of a reactive ester,preferably with N-hydroxy succinimide (NHS), to form LDPEG-SS, and thenreacting of LDPEG-SS with an accessible reactive site on SOD, preferablya primary amine residue on SOD, mainly lysine epsilon amine.

Referring specifically to LDPEG-SOD, the process is as shown: ##STR1##wherein: LDPEG--OH=low diol CH₃ O--PEG--OH containing not more thanabout 10% w/w of HO--PEG--OH

LDPEG--SS=low diol CH₃ O--PEG--OCOCH₂ CH₂ COO(C₄ H₄ NO₂) containing notmore than 10% of [C₄ H₄ O₂ N)OOC--CH₂ CH₂ COO]₂ PEG

LDPEG--S=low diol CH₃ O--PEG--OCOCH₂ CH₂ COOH containing not more than10% of [HOOC--CH₂ CH₂ --COO]₂ PEG

DCC=dicyclohexylcarbodiimide

SA=succinic anhydride

bSOD=Bovine Superoxide Dismutase

NHS=(C₄ H₄ NO₂)OH, N-hydroxysuccinimide

(LDPEG)_(n) bSOD=low diol(CH₃ O--PEG--OCOCH₂ CH₂ CO)_(n) --bSOD

(LDPEG)_(n-1) bSOD--S=low diol(CH₃ O--PEG--OCOCH₂ CH₂ CO)_(n-1)--bSOD--COCH₂ CH₂ COOH

SAcid=Succinic Acid

n=number of low diol PEGs per SOD

K₁, K_(obs), k₂ and k₃ are rate constants for the reactions.

The process of attaching LDPEG to catalase is analagous to that ofattaching LDPEG to SOD.

Essential components used in the formulations of the present inventionwill now be described.

Starting Materials, Intermediates and Reagents

Superoxide Dismutase

Superoxide dismutase is the name given to a class of enzymes thatcatalyze the breakdown of the superoxide anion radical (O₂ ⁻.) to oxygenand hydrogen peroxide.

SOD is known under the systematic nomenclature of the InternationalUnion of Biochemistry as superoxide oxidoreductase and has aclassification number of 1.15.1.1. Such substances have been calledorgoteins and hemocupreins as well as superoxide dismutases and range inmolecular weight from about 4,000 to about 48,000. The copper-zincdismutases are a remarkably conserved family with respect to grossstructural properties. Without exception, the purified enzymes have beenshown to be dimers (molecular weight usually 31,000-33,000) containingtwo moles each of copper and zinc ions per mole. The enzymes of themanganese/iron family are not as uniform in such basic properties asmolecular weight, subunit structure and metal content. Some are dimers;others are tetramers. The content of metal ranges from about 0.5 to 1mole per mole of subunit polypeptide chain. Naturally occurringZn/Cu-containing enzymes from mammals and their functionally competentanalogs and muteins are considered to be mammalian Zn/Cu superoxidedismutases (mSOD).

In formulations of the present invention mSOD may be of any origin. Itis commercially obtained from bovine erythrocytes and human erythrocytesas well as by recombinant synthesis in microorganisms, such as E-coliand yeast. Among other sources, Cupri-Zinc bovine liver superoxidedismutase (SOD, EC 1.15.1.1) for example, is available from DDIPharmaceuticals, Inc. (Mountain View, Calif.).

Catalase

Catalase is an antioxidant enzyme that specifically catalyzes thedecomposition of hydrogen peroxide to water and oxygen. It is found inanimals, plants, fungi and certain bacteria. Catalase is commerciallyavailable isolated from animal livers (bovine hepatocatalase) andkidneys as well as bacteria (Micrococcus Lysodeikticus) and fungi(Aspergillus Niger). All catalases so far isolated have an approximatemolecular weight of 240,000 daltons.

Catalase is extensively used in industrial food production for detectingresidual catalase activity in processed and preserved foods whereperoxidatic reactions effect changes of color, taste and odor of thefood. In the dairy industry catalase is used as an easily detectableindicator of contamination of milk. Because of its protective propertyof being an H₂ O₂ -scavenger, it is proposed for use in reperfusioninjury following ischemia in myocardial infarction and stroke, burns,trauma, renal transplants, respiratory distress syndrome andbroncho-pulmonary displasia.

Polyethylene Glycol

In practicing the present invention, we prefer to use low diol PEG forattachment to biologically active proteins. Polyethylene glycol is alinear, hydrophilic, uncharged, non-immunogenic molecule which isreadily excreted in the urine following ingestion or injection.Polyethylene glycol exists as a mixture of two forms:

one form contains one --OH group CH₃ (OCH₂ CH₂)_(n) OH, calledmono-methoxylated polyethylene glycol since it contains one methoxygroup per molecule of polyethylene glycol; and

the other form contains two --OH groups H(OCH₂ CH₂)_(n) OH, called the"diol" form of polyethylene glycol since it contains two --OH groups perone molecule of polyethylene glycol.

The chain-length of the polyethylene glycol and its molecular weightdepends on the magnitude of n: the larger the n, the higher themolecular weight.

While certain molecular weight methoxypolyethylene glycols are availablecommercially (for example, methoxy-PEG₅,000 was obtained from UnionCarbide Corporation in two forms: a conventionally available high diolmethoxy-PEG₅,000 which contained 14-17% of higher molecular weight PEGdiol, and a low diol product which contained less than 4% PEG diol) someare required to be made and purified in order to produce a pegatedprotein that possesses low immunogenicity. For example, pegation of SODwith methoxy-PEG-SS derived from some commercial sources leads to aproduct containing high molecular weight components, as verified by sizeexclusion chromatography, discussed earlier. This high molecular weightproduct is believed to derive from protein crosslinking through anactivated diester formed from the various amounts of PEG diol found inthe commercial sources of M-PEG. The individual active esters, althoughlocated on the same polymer chain, are nonetheless chemically remotefrom one another. Thus, the presence of a second reactive functionalityin the polymer tends to exert an increasingly negligible effect on thereactivity of a first reactive functionality as the distance separatingthe two functionalities increases. The individual reactivities thus tendto be independent of moieties present at opposite ends of the polymerchain, and crosslinking cannot be avoided in the absence of infinitedilution of reagents. It is, accordingly, important to synthesize anM-PEG-SS known to contain very small amounts, preferably no amounts ofdiester. S. Zalipsky et al in the Journal of Bioactive and CompatiblePolymers, Vol. 5, April 1990, pp. 227-231, described the purification ofpolyethylene glycol 2000 from methoxypolyethylene glycol 2000. Thesuccinate esters are also prepared and shown to separate by ion exchangechromatography on DEAE-Sephadex. The preparative method is shown inExample 3.

While the procedure described in Example 4 works well with PEG-2000, itfails with higher molecular weight PEG's. Higher molecular weight PEGacids do not bind to anion or cation resins; the greater mass ofpolyethylene backbone is believed to mask any ionic properties of thependant acid. We have found that extremely low ionic strength buffer wasrequired to bind the PEG succinates and they eluted under very lowincreases of ionic strength indicating that they are only very weaklyheld by the resin.

We have found that higher molecular weight methoxy-PEGs can be separatedfrom diol components if the hydroxyl functionalities are first convertedto dimethoxytrityl (DMT) ethers before application of reverse phase thinlayer chromatography. The hydroxyls can be liberated by acid treatment.

The schematics of preparation and purification of methoxy-PEG₅₀₀₀-dimethoxytrityl (M-PEG-DMT) derivatives are as follows; while thedetails are shown in Examples 4 through 7.

M-PEG-DMT and DMT-PEG-DMT are prepared in an identical fashion. Thepolyether is dissolved in ethanol-free chloroform and the solution driedby distilling off approximately half the chloroform at atmosphericpressure under a blanket of argon. The solution is then allowed to coolto room temperature under argon, followed by the sequential addition ofexcess diisopropylethyl amine (1.5 eq.), 10 mol %4-dimethylaminopyridine as catalyst, and finally an excess amount of4,4-dimethoxytrityl chloride (1.2 eq.). After 15 hours reaction, thesolution is concentrated by rotary evaporation and the solution added toanhydrous ether to precipitate the tritylated PEG. Regular phase TLCcleanly separates starting material from product, the PEG backbonestaining with Dragendorf's reagent. While M-PEG-DMT is not resolved fromDMT-PEG-DMT by regular phase TLC, reverse phase C-18 TLC plates cleanlyseparate M-PEG-DMT, DMT-PEG-DMT, DMT chloride and DMT alcohol from eachother (mobile phase 4:1:1 acetonitrile/water/isopropanol). PEG backboneis confirmed by staining orange to Dragendorfs and trityl incorporationconfirmed by exposing the plate to HCl vapors to give an orange stain.

Authentic M-PEG-DMT 5000 was shown to separate cleanly from authenticDMT-PEG-DMT 8000 on a Waters C-8, 300 angstrom pore size, 15-20 micronparticle size Prep-Pak Bondapak cartridge. The crude M-PEG-DMT wasdissolved by sonication in 30% acetonitrile/water to a concentration ofapproximately 12 mg/ml and passed through a 2.5 micron filter. Thesample was loaded onto the column (2 g in 25 ml) in a 30%acetonitrile/water mobile phase. After 8 minutes of isocratic elution, acontaminating peak eluted (identity unknown, having a high absorbance at280 nm but accounting for very low relative mass). A gradient of 30-70%acetonitrile/water over 21 minutes was then begun, and the desiredM-PEG-DMT eluted at 58-60% acetonitrile. Authentic DMT-PEG-DMT typicallyelutes at 80% acetonitrile. The first 3/4 of the desired peak iscollected and the last 1/4 discarded. In this way, 15.4 g of M-PEG-DMTwas purified from 22.6 g of crude M-PEG-DMT.

The trityl cleavage of M-PEG-DMT is as follows:

attempted removal of the DMT group from M-PEG-DMT with HCl gave by TLC(crude undiluted reaction mixture) complete removal of the trityl group.However, concentration of the chloroform extract gave a back reactionwhich resulted in a re-tritylation of a significant portion of the PEG.It was not possible to purify this by selective precipitation. Thehydrated trityl cation and chloride are apparently in equilibrium withthe result that dehydration, such as occurs during solvent removal,produces significant quantifies of DMT chloride. This re-tritylation maybe prevented by the use of a non-equilibrating counterion. Sulfuric acidwas shown to irreversibly de-tritylate M-PEG-DMT. The sulfuric acidcleaved M-PEG is extracted into chloroform, concentrated andprecipitated into ether to give pure zero diol M-PEG. In this manner, 10g of M-PEG-DMT was cleaved to 8.68 g of zero diol M-PEG. Size exclusionchromatography indicates this material contains less than 0.3% diol.

Other higher molecular weight methoxy-PEG derivatives can be made byanalogous processes.

The following examples will serve better to illustrate preparation ofthe low diol PEG/proteinaceous conjugates to form a complex withcyclodextrin.

EXAMPLE 1 A. Methoxypolyethylene Glycol Succinate (M-PEG-S)

In a 2 liter flask, 100 g (0.02 mole) of methoxy-PEG₅,000 (M-PEG) wasdissolved with stirring in 300 ml of warm (40° C.) anhydrous toluene.The volume was reduced by azeotropic removal of 147 ml of toluene undera nitrogen atmosphere to reduce the water content of the m-PEG from 1.73to 0.23%. After cooling to ambient temperature, 233 ml of dry methylenechloride followed by 3.0 g (0.09 moles) of succinic anhydride and 1.1 g(0.01 mole) of 4-dimethylaminopyridine (DMAP) were added. The reactionwas stirred and heated at reflux overnight, and then 200 ml of methylenechloride was removed at reduced pressure. The residue was added withstirring to 1.6 liters of ether in a 4 liter flask. This was stirred for45 minutes and filtered. The filter cake was washed with 70 ml of etherand dried at reduced pressure to afford 100.4 g of crude m-PEG-succinate(m-PEG-S) as a white solid containing DMAP.

The crude M-PEG-S (100 g) was dissolved in 633 ml of methylene chlorideand passed through a column containing 114 g of Dowex 50×8-100H+ resinpreviously washed with 272 ml di dry methylene chloride. The column wasthen washed with an additional 316 ml of methylene chloride, and theeluents were combined and dried over anhydrous magnesium sulfate.Methylene chloride (800 ml) was removed under reduced pressure. Theremaining solution was added with stirring to 1600 ml of ether in a 4000ml flask. After stirring for 30 minutes, the suspension was allowed tostand for 30 minutes and then filtered. The filter cake was then washedwith 75 ml of ether and dried at reduced pressure. This afforded 96.0 g(94% yield) of m-PEG-S as a white solid which exhibited a proton NMRspectrum consistent with the assigned structure: ¹ H-NMR (CDCl₃): 4.27(triplet, 2H, --CH₂ --O--C(═O)--), 3.68 (large singlet offscale, PEGmethylene O--CH₂ --'s), 3.39 (singlet, 3H, OCH₃), and 2.65 ppm (narrowmultiplet, 4H, --C(═O)--CHhd 2--CH₂ --C(═O)--). The carboxylic acidcontent of 0.000207 mol/g was measured by titration.

B. Methoxypolyethylene Glycol N-Succinimidyl Succinate (M-PEG-SS)

In a 2,000 ml flask, 98.48 g (0.0192 mole) of methoxypolyethylene glycolsuccinate (m-PEG-S) was dissolved in 468 ml of dry toluene with warmingto 40° C. The solution was filtered and the volume was reduced by 263 mlby azeotropic distillation under nitrogen. The resultant viscous liquidwas transferred to a 1,000 ml three-necked flask under nitrogen using225 ml of dry methylene chloride. To this was added 2.22 g (0.0192 mole)of N-hydroxysuccinimide, and the reaction was stirred until theN-hydroxysuccinimide dissolved. The reaction mixture was then cooled to5° C. in an ice bath, and a solution of 4.44 g (0.0125 mole) ofdicyclohexylcarbodiimide (DCC) in 24 ml of methylene chloride was addeddropwise over 5 minutes. During the addition of the methylenechloride/DCC solution, dicyclohexylurea (DCU) began to crystallize fromthe reaction mixture. The reaction was allowed to warm to roomtemperature and was stirred overnight. The content of the reaction flaskwas transferred to a 2,000 ml flask using 25 ml of methylene chloride torinse the flask. At reduced pressure at 30° C., 250 ml of methylenechloride was removed, the suspension was filtered and the filter cakewas washed with 25 ml of dry toluene. The filtrate was then added to1,200 ml of anhydrous ether with stirring, and the resultant suspensionwas stirred for 45 minutes before being filtered. The filter cake wasrinsed with 100 ml of dry ether and dried under a latex rubber dam for 2hours. The resultant solid was then dried under high vacuum andtransferred to a bottle in a glove bag under argon. This afforded 96.13g (96.1% yield) of the title compound (m-PEG-SS) as a white solid whichexhibited a proton NMR spectrum consistent with the assigned structure:¹ H-NMR (CDCl₃): 4.32 (triplet, 2H, --CH₂ --O--C(═O)--), 3.68 (largesinglet offscale, PEG methylene O--CH₂ --'s), 3.39 (singlet, 3H, OCH₃),2.99 and 2.80 (pair of triplets, each 2H, succinate --C(═O)--CH₂ CH₂--C(═O)--), and 2.85 ppm (singlet, 4H, succinimide --C(═O)--CH₂ CH₂--C(═O)--). The active ester content of the product was determined byreaction with excess benzylamine in toluene followed by back titrationwith perchloric acid in dioxane to a methyl red end-point and found tobe 0.000182 mole/g.

C. Low Diol PEG-SOD

11.8 g of an aqueous solution of SOD containing 82.1 mg of protein pergram was diluted to a total weight of 200 g with 0.1M sodium phosphatebuffer at pH7.8. To this solution, magnetically stirred and heated to30° C., was added 9 3.4 g of low diol methoxy PEG-SS prepared inExample 1. The pH of the reaction mixture was maintained at 7.8 using aMettier DL25 titrator programmed in the pH stat mode to add 0.5 normalsodium hydroxide solution as required. After 1 hour the reaction mixturewas filtered through a 0.2 micron low protein binding polysulfonefilter, concentrated to about 60 ml using a stainless steel MilliporeMini-tan device equipped with a 30,000 NMWL membrane 4 pk and was thensubjected to dialfiltration against 2 liters of 50 mM sodium phosphatebuffered saline (0.85%) at pH 6.2 to 6.3. The retentate solutioncontaining the low diol PEG-SOD was then filtered through a 0.2 micronfilter.

EXAMPLE 2 A. Monomethoxypolyethylene Glycol Succinate

A 12 liter three-neck flask was charged with 4 liters of toluene and2212 g of methoxypolyethylene glycol, previously warmed to 70° C. undernitrogen. The volume was reduced by azeotropically removing 1.3 litersof toluene at reduced pressure. After cooling to 30° C., there was added4 liters of methylene chloride followed by 66.4 g of succinic anhydrideand 24.4 g of 4-dimethylaminopyridine. The reaction was refluxed for 32hours then 3.8 liters of methylene chloride was removed at atmosphericpressure. The reaction was cooled and poured into a 5 gal. glass carboycontaining 28 liters of methyl tert-butyl ether with stirring. Theresulting suspension was stirred for 1 hour and collected on a Lappfilter. The filter cake was washed with 1 liter of methyl tert-butylether. Drying in a vacuum over overnight at room temperature yielded2.252 kg of the title compound as a crude white solid.

The crude title compound was dissolved in 8 liters of methylene chlorideand passed through a glass pressure column containing 3.0 kg of Dowex50W-X8 resin (cation exchange, hydrogen form) previously washed with 5liters acetone followed by 4 liters of methylene chloride. The columnwas then washed with 3 liters of methylene chloride. The column eluentswere combined and 10 liters of methylene chloride was removed atatmospheric pressure. The remaining solution was poured into 26 litersof methyl tert-butyl ether with stirring. The resulting suspension wasstirred for 45 minutes and the solid was removed by filtration. This waswashed with 3 liters of methyl tert-butyl ether. Drying in a vacuum ovenat room temperature yielded 2.46 kg of a white solid of the titlecompound, 95% recovery. This material contained 1.5% methoxypolyethyleneglycol, and assayed at 2.72×10⁻⁴ mole/g (theory is 1.96×10⁻⁴ mole/g).

B. Methoxypolyethylene Glycol N-Succinimidyl Succinate

In a 12 liter flask under nitrogen 1.5 kg of monomethoxypoly ethyleneglycol succinate was dissolved in 7.2 liters of toluene with warming.The volume was reduced by 2.8 liters at reduced pressure to removewater. The resultant viscous liquid was cooled to 40°-45° C. and 3.4liters of methylene chloride was added followed by 33.89 g ofN-hydroxysuccinimide. The reaction was stirred for 1 hour until all theN-hydroxysuccinimide was dissolved, then the reaction was cooled to 10°C. and a methylene chloride solution (368 ml) of 67.75 g1,3-dicyclohexylcarbodiimide (DCC) was added dropwise over 30 minutes.The reaction was allowed to warm slowly to room temperature while beingstirred over 18 hours. The volume was then reduced by 3.2 liters atatmospheric pressure. The suspension was cooled to 0°-5° C. and stirredfor 30 minutes. This was filtered and the filter cake was washed with250 ml of toluene. The filtrate and the wash was added to 28 liters ofmethyl tert-butyl ether with stirring. The resultant suspension wasstirred for 45 minutes and then filtered on a Lapp filter. The filtercake was washed with 1 liter of methyl tert-butyl ether and dried undera latex dam for 4 hours. Additional drying at room temperature in avacuum oven at reduced pressure overnight yielded 1.5 kg of a whitesolid, 100% yield. This material assayed at 1.79×10⁻⁴ mole/g (theory is1.92×10⁻⁴ mole/g).

C. Methoxypolyethylene Glycol Succinoyl Bovine Superoxide Dismutase

To 32 liters of warm (29°-30° C.) pH 7.8 phosphate buffer in a 42 litersreactor containing a pH electrode was added 194.0 g of bovineerythrocyte superoxide dismutase. The volume was adjusted to 39.5 litersand the reaction was warmed to 29° C. The sodium hydroxide tube from thepH titrator was adjusted over the center of the reactor directly abovethe surface of the solution. The pH titrator was initiated and the pHwas adjusted to 7.8 with 0.5N sodium hydroxide. At this time 614.7 g ofmethoxypolyethylene glycol N-succinimidyl succinate was added over twominutes and the reaction was stirred for 41 minutes while the pH wasbeing adjusted to 7.8 with 0.5N sodium hydroxide with the reactiontemperature being maintained at 30° C. The reaction was then filteredthrough a 200 Millipak filter and concentrated using a Milliporestainless steel Pellicon diafiltration system. The reactor was thenrinsed with 600 ml of pH 6.2 phosphate buffer. The rinse was added tothe concentrate after filtering through the Millipore 200 filter and thedialfiltration system. The final volume of the concentrate was about 9liters. The concentrate was then diafiltered, using the MilliporePellicon diafiltration system against 200 liters of pH 6.2 phosphatebuffer over 2.17 hours. The diafiltration system was rinsed with 1.5liters of pH 6.2 phosphate buffer. The final volume of the concentratewas about 8 liters. The concentrate was then transferred to a clean 5gal glass carboy through an inline Millipore 200 Millipak filter and thefilter was rinsed with 500 ml of pH 6.2 phosphate buffer. This afforded11.98 kg (91.4% yield) of the title compound as a clear greenish-bluesolution. (Activity: 32,960 units/ml).

EXAMPLE 3 A. Preparation of Partially Carboxymethylated PolyethyleneOxide

Polyethylene oxide, M_(w).spsb.2000 (Fluka, 25 g, 25 meq. OH) wasdissolved in toluene (120 ml) and azeotropically dried until no morewater appeared in the Dean-Stark trap attachment (approx. 25 ml oftoluene were removed). The solution was cooled to 50° C. and treatedwith potassium tert-butoxide (1.7 g, 15 mmol). The solution was broughtto reflux and more solvent was distilled off (approx. 25 ml). Thestirred reaction mixture was brought to 25° C., and treated overnightwith ethyl bromoacetate (3.4 ml, 16 mmol). The precipitated salts wereremoved by gravity filtration, and washed with methylene chloride (30ml). The polymer was recovered by partially concentrating the filtrate(to approx. 60 ml), and slowly pouring the concentrated solution intoethyl ether (300 ml) at 5° C. with vigorous stirring. The collectedwhite polymeric powder was dried in vacuo. Yield: 24 g; IR (neat) showedthe characteristic ester absorption at 1753 cm⁻¹. The polymer wasdissolved in 1N NaOH (50 ml), and NaCl (10 g) was added. After approx.45 rain this solution was acidified with 6N HCl to pH 3.0 and extractedwith methylene chloride (3×60 ml). The combined organic phases weredried (MgSO₄), concentrated (to approx. 50 ml), and poured into coldstirring ether (300 ml). The precipitated product was collected byfiltration and dried in vacuo. Yield: 22 g; IR (neat) showed absorptionat 1730 cm⁻¹, corresponding to ω-carboxyl group.

B. Preparation of Pure α-Hydroxy-ω-Carboxymethylpolyethylene Oxide bySeparation of Partially Carboxymethylated PEO on DEAE-Sephadex

The mixture of homo- and heterobifunctional PEO's (22 g) was dissolvedin water (40 ml), and applied to a column containing DEAE-Sephadex A-25(Sigma, 27 g, 0.1 mole ion-exchange sites) in the tetraborate form. Thefirst fraction containing underivatized polymer was eluted withdeionized water. When the eluent became negative to a PAA test, astepwise ionic gradient of ammonium bicarbonate (from 6 to 22 mM atincrements of 1-2 mM every 100 ml) was applied, and fraction collection(approx. 40 ml each) began. Fractions 2-21 were positive to the PAAtest, and contained pure monocarboxylated PEO (R₁ =0.49). The subsequentthree fractions did not contain PEO, while fractions 25-36 contained thepure PEO-diacid (R₁ =0.26). The fractions containingα-hydroxy-ω-carboxymethylpolyethylene oxide were combined andconcentrated (to approx. 100 ml). Sodium chloride (35 g) was dissolvedin this solution, which was then acidified to pH 3 and extracted withmethylene chloride (3×100 ml). The combined CH₂ Cl₂ solution was dried(MgSO₄), concentrated (to approx. 100 ml), and slowly poured into coldstirring ether (500 ml). The precipitated polymer was collected andthoroughly dried in vacuo to give 8.8 g of product. ¹³ C-NMR (CDCl₃):δ172.7 (COOH); 72.4 (CH₂ CH₂ OH); 70.4 (PEO); 69.0 (CH₂ COOH); 61.3 (CH₂OH) ppm.

Bis-carboxymethylpolyethylene oxide isolated from the column was alsoanalyzed. ¹³ C-NMR (CDCl₃): δ172.4 (COOH); 70.4 (PEO); 68.8 (CH₂ COOH)ppm.

EXAMPLE 4 Synthesis of Dimethoxytrityl Methoxypolyethylene Glycol

Methoxypolyethylene glycol (5,000 dalton average molecular weight; 36.3g, 7.26 mmol) was dissolved in 500 ml chloroform, followed by theremoval by distillation of 250 ml chloroform to remove water. A dryingtube was attached to the flask and the solution allowed to cool toapproximately 50° C. N,N-diisopropylethylamine (1.8 ml, 10.3 mmol) wasadded, followed by 4-dimethylamino pyridine (100 mg, 0.8 mmol, 10 mol %)and 2.9 g of 4,4-dimethoxytrityl chloride (98%).

The mix was allowed to stir overnight at room temperature at which timethe solvent was removed by rotary evaporation at 60° C. The residue wastaken up in a small amount of chloroform, and the M-PEG-DMT wasprecipitated by addition into 2 liters of anhydrous ether. Theprecipatate was collected, dried and chromatographed on a C-8 300Areverse phase prep column on a Waters LC4000 system employing a 30-95%acetonitrile gradient (against water) over 20 minutes. The desiredproduct eluted at 58-60% acetonitrile. The sample (2 g) in 20 ml of 30%acetonitrile/water was loaded onto the column at 50 ml/min flow rate.This eluent (30% acetonitrile/water) was allowed to continueisocratically until a large impurity peak was eluted, typically 3-5 min,mv 280 μm. After the elution of this first peak, the gradient wasstarted. The next peak to elute was the desired methoxy-PEG-DMT 5000.The first 3/4 of the peak was collected, and the tail end of the peakwas discarded.

In this fashion, 22.6 g of crude M-PEG-DMT 5000 was purified in 2 gportions to obtain 15.44 g of the title product.

EXAMPLE 5 Synthesis of Zero Diol Methoxypolyethylene Glycol fromDimethoxytrityl Methoxypolyethylene Glycol

10 g M-PEG-DMT 5000 was placed in a 500 ml flask and dissolved in 320 mlMilli-Q water. Sulfuric acid was added (80 ml) as a slow stream to bringthe concentration to 20%. The solution turned red and homogeneous. Afterstirring overnight, the acid solution was extracted with 2×500 mlchloroform, and the combined extracts dried over MgSO₄, concentrated,and the red oil poured as a thin stream into 2 liters of anhydrous etherat 20° C. The precipitate was allowed to settle for 24 hours. It wascollected in a course frit sintered glass funnel, and then washed with2×200 ml portions of anhydrous ether. The precipitate cake was broken upand dried under vacuum to yield 8.68 g methoxy-PEG 5000 (zero diol).

EXAMPLE 6 Synthesis of Zero Diol Methoxypolyethylene Glycol Succinatefrom Zero Diol Methoxypolyethylene Glycol

M-PEG-OH 5000 zero diol (4.7 g, 0.94 mmol) was dissolved in 100 mltoluene. The solution was brought to reflux and a Dean-Stark trap wasused to remove any water. After 1 hour at reflux, a total of 80 mltoluene was removed by distillation, and the vessel containing 20 mltoluene, was allowed to cool under argon positive pressure. Succinicanhydride was added (110 mg, 1.1 mmol), followed by4-dimethylaminopyridine (137 mg, 1.12 mmol). Since the succinicanhydride did not dissolve, 10 ml of anhydrous ethanol free chloroformwas added, and the solution was held at a reflux using an oven driedcondenser. After 15 h at reflux, the solution was cooled and thenstirred with 10 g of cation exchange resin, filtered, and the filtrateconcentrated to obtain the title compound.

EXAMPLE 7 Synthesis of Zero Diol Methoxypolyethylene Glycol SuccinimidylSuccinate Zero Diol Methoxypolyethylene Glycol Succinate

A solution of M-PEG-succinate from Example 7 (4.15 g, 0.83 mmol) in 100ml of toluene was dried azeotropically. A portion of the toluene wasdistilled off (60 ml, leaving 40 ml in the reaction flask) andN-hydroxysuccinimide (100 mg, 0.87 mmol) was added, followed by thecareful addition of 30 ml of ethanol free anhydrous chloroform. Anadditional 25 ml of the mixed solvent was removed by distillation andthe solution was allowed to cool at room temperature under argon. DCCwas added (200 mg, 9.7 mmol) and the solution was stirred. After 10minutes, DCU began to crystallize out. After stirring for two days, anadditional 25 mg (0.22 mmol) of N-hydroxy succinimide was added. Thedicyclohexyl urea (DCU) slurry was filtered and the precipitate waswashed with toluene. The filtrate was concentrated by rotary evaporationgiving an additional precipitation of dicyclohexyl urea (DCU). Thefiltered concentrate was added dropwise into one liter of anhydrousether. The precipitate was collected on a Whatman 9 cm 6F/F glass fiberfilter and then dried under high vacuum for 15 hours, to give 3.37 g ofM-PEG-SS.

Active ester content: 1.71×10⁻⁴ mol/g; HPLC indicated: 1.3% M-PEG-S;other impurities: 1.2%; DCU none detected; total impurity: 3%.

EXAMPLE 8 Synthesis of Zero Diol PEG-SOD

Superoxide dismutase (1.33 ml of 75 mg/ml stock) was added to 18.67 mlof reaction buffer (100 mM sodium phosphate, pH 7.8) and the solutionwas brought to 30° C. M-PEG-SS from Example 8 (300 mg) was added in oneportion and the pH was maintained at 7.8 by use of a pH stat. After 28minutes the reaction pH became unchanging and the sample wasconcentrated on Centrium centrifugal membrane of 10,000 MW cutoff. Theconcentrated sample was exchanged in this manner with Dulbecco's PBSwhich had been adjusted to pH 6.2 with 1M HCl. Five exchanges at a totalof 60 ml were performed. Size exclusion HPLC showed negligable high MWpeak indicating that the title compound contained negligable amounts ofmaterial derived from diol (i.e., it was "zero diol").

The following examples illustrate the preparation of other biologicallyactive proteins covalently joined to PEG.

EXAMPLE 9 Synthesis of Low Diol MethoxypolyethyleneGlycol-Succinoyl-Catalase

4.17 ml of an aqueous suspension of catalase containing 24.0 mg ofprotein per ml was diluted with 15.84 ml of 0.1M sodium phosphatebuffer, pH 7.8. To this solution, magnetically stirred and heated to 30°C., was added 550 mg of low diol methoxy PEG-SS. The pH of the reactionmixture was maintained at 7.8 using a Mettier DL25 titrator programmedin the pH stat mode to add 0.5 normal sodium hydroxide solution asrequired. After 0.5 hour the reaction mixture was filtered through a0.45 micron low protein binding polysulfone filter and placed in twoAmicon Centriprep 30 Concentrators (30K NMWL membrane) and buffer wasexchanged several times with Dulbecco's PBS. The retentate solutioncontaining the low diol PEG-catalase was then filtered through a 0.2micron filter. Conjugate formation was demonstrated by SEHPLC and gelelectrophoresis.

EXAMPLE 10 Synthesis of Low Diol PEG-Ovalbumin

503 mg of ovalbumin (Sigma) was dissolved in 50 g of 0.25M, pH 7.8phosphate buffer at room temperature in a polyethylene beaker containinga Telfon-coated magnetic stir bar. After stirring for 15 minutes, 1.900g of low diol M-PEG(5,000)-SS was added all at once. The pH of thereaction mixture was controlled at 7.8 with a Mettier DL25 pH stat whichadded 0.5N NaOH as needed. The reaction was allowed to continue for 1 hat room temperature, and then the reaction mixture was diafilteredthrough an Amicon YM30 membrane using a stirred cell device operatedunder 25 psi of argon overnight in a refrigerator at 4° C. After 800 mlof buffer had beed diafiltered, the product was concentrated byultrafiltration, filtered through a 0.2 micron polysulfone filter, andvialed in sterile glass vials to give 44.3 g of solution with a proteincontent of 10.5 mg/ml. The degree of protein modification was determinedto be 71.4% by titration analysis of lysine amines.

EXAMPLE 11 Synthesis of Low Diol mPEG_(5K) -S-Ovalbumin

10 ml of a cold 10 mg/ml solution of ovalbumin (Sigma, grade VI) in0.25M phosphate buffer, pH 7.4 was added to 382 mg of low diol mPEG_(5K)-SS and stirred at 5° C. for 16 hours. The product was purified in aCentriprep 30 Concentrator (Amicon, 30K NMWL membrane) using Dulbecco'sPBS as the exchange buffer. The purified solution was filtered through a0.2 μm filter to give 6.539 g containing 13.8 mg/ml of 74% modified(TNBS titration method) protein.

In a similar manner, 100 mg of ovalbumin was reacted with 283 mg of lowdiol mPEG_(5K) -SS giving 6.274 g containing 13.8 mg/ml of 74% modifiedprotein.

In a similar manner, 100 mg of ovalbumin was reacted with 190 mg of lowdiol mPEG_(5K) -SS giving 5.704 g containing 16.8 mg/ml of 67% modifiedprotein.

EXAMPLE 12 Synthesis of Low Diol mPEG_(5k) -S-rhu-IL4

190 μl of a 5.26 mg/ml solution of rhu-IL4 (Immunex) was diluted with772 μl of 0.1M borate buffer, pH 8.5. The rhu-IL4 solution was thentreated with 29.2 μl of a 34 mg/ml solution of low diol methoxy PEG-SSin DMF. After 1 hour and 20 minutes at room temperature the reactionmixture was centrifuged and injected directly onto a preparative SEHPLCcolumn. The purified conjugate was shown to be essentially a single bandon gel electrophoresis.

EXAMPLE 13 Synthesis of Low Diol mPEG_(5K) -S-NT

A solution containing 8.7 mg of neurotensin (NT) (BaChem) in 2.175 ml of0.25M phosphate buffer, pH 7.8 was added to 174 mg of low diol mPEG_(5K)-SS. The reaction mixture was kept at room temperature for 1.75 hours,then refrigerated. Pure mono-mPEG_(5K) -S-NT was obtained afterseparation from NT-PEG_(8K) -NT by preparative reverse phase HPLC on aC-8 column eluting with a water/acetonitrile gradient.

The formulations of the present invention are made using conventionaltechniques known to those skilled in the art. Optionally, theformulation may contain sucrose or trehalose.

Specific formulations are illustrated in Tables I and II.

                  TABLE I                                                         ______________________________________                                                PEG SOD   Phosphate                                                   Example (U/ml)    Buffer (mM) pH  HPCD % w/v                                  ______________________________________                                        14      25,000    10          6.2  5                                          15      50,000    10          6.2  5                                          16      75,000    10          6.2  5                                          17      100,000   10          6.2  5                                          18      125,000   10          6.2  5                                          19      150,000   10          6.2  5                                          20      25,000    10          6.2 10                                          21      50,000    10          6.2 10                                          22      75,000    10          6.2 10                                          23      100,000   10          6.2 10                                          24      125,000   10          6.2 10                                          25      150,000   10          6.2 10                                          26      25,000    10          6.2 20                                          27      50,000    10          6.2 20                                          28      75,000    10          6.2 20                                          29      100,000   10          6.2 20                                          30      125,000   10          6.2 20                                          31      150,000   10          6.2 20                                          ______________________________________                                    

                  TABLE II                                                        ______________________________________                                                PEG       Phosphate                                                   Example Catalase  Buffer (mM) pH  HPCD % w/v                                  ______________________________________                                        32      25,000    10          6.2  5                                          33      50,000    10          6.2  5                                          34      75,000    10          6.2  5                                          35      100,000   10          6.2  5                                          36      125,000   10          6.2  5                                          37      150,000   10          6.2  5                                          38      25,000    10          6.2 10                                          39      50,000    10          6.2 10                                          40      75,000    10          6.2 10                                          41      100,000   10          6.2 10                                          42      125,000   10          6.2 10                                          43      150,000   10          6.2 10                                          44      25,000    10          6.2 15                                          45      50,000    10          6.2 15                                          46      75,000    10          6.2 15                                          47      100,000   10          6.2 15                                          48      125,000   10          6.2 15                                          49      150,000   10          6.2 15                                          50      25,000    10          6.2 20                                          51      50,000    10          6.2 20                                          52      75,000    10          6.2 20                                          53      100,000   10          6.2 20                                          54      125,000   10          6.2 20                                          55      150,000   10          6.2 20                                          ______________________________________                                    

Formulations of the present invention are lyophilized using thefollowing process.

Vials of required dimensions are chosen to be filled by a formulationbased upon dose requirements. In choosing vials to accommodate a dose,the fill volume should not exceed the diameter of the vial. For example,a 5 ml fill should not be introduced into less than a 10 ml vial. Afterfilling, the vials are loaded into the drying chamber and placeddirectly onto the refrigerated shelves which were pre-chilled to 4° C.Thermocouples are placed inside a number of the vials to monitor thetemperature of the formulation during the lyophilization process. Thevials are then allowed to equilibriate to the temperature of the shelves(4° C.) before lowering the shelves' temperature to -40° C. Oncereaching -40° C., the vials are kept at this temperature for about 6 hrsto allow complete freezing of the formulation. After this time periodthe condenser coils are chilled to -80° C. and the vacuum pump is turnedon to evacuate the condenser chamber followed by the process of primaryand secondary drying. In the primary drying process, the main valvebetween the condenser and the drying chamber is opened and the dryingchamber is evacuated to a pressure of about 100 microns with a nitrogengas bleed. Upon reaching a pressure of 100 microns, the shelftemperature is raised to -20° C. to start the sublimation process. Thisportion of the lyophilization cycle requires about 10 to 18 hrs. Theprimary drying process is complete when all of the ice disappears fromthe frozen matrix and the thermocouple temperature has reached -20° C.In the secondary drying process, the temperature is raised from -20° C.to +25° C. to remove all the ice that was not removed during thelyophilization process. This removal required approximately 4 to 8 hrs.

After the completion of the secondary drying process the main valve isclosed off and the drying chamber is filled with nitrogen so as tomaintain a slight vacuum in the chamber. The stoppering ram is thenactivated and the closures are pushed down into the vials. The dryingchamber is then equilibriated to atmospheric pressure and the chamberdoor is opened to remove the vials and apply the crimp seals. The vialsthen are stored at the prescribed temperature until reconstituted withwater for injection.

Shelf-life of the formulations of the present invention were found to beexcellent. Shelf-life is illustrated by the following comparativestudies using reconstituted solutions to observe visual appearance ofthe formulations and Size Exclusion High Performance LiquidChromatography (SEHPLC) to determine % high molecular weight (HMW) ofmaterial contained in the formulations shown in Examples 56 and 57.

Formulation A, according to the present invention, contained 10% HPCD.

Formulation B, for comparative purposes, contained 10% sucrose and noHPCD.

Both formulations contained 10 mM phosphate buffer at pH 6.2 and 75,000U/ml PEG SOD. Fill volume of vials was 0.5 ml; temperature conditionswere 4° C., 22° C., 30° C. and 50° C.; both formulations werelyophilized and maintained at the above temperatures for 30 months, andthen reconstituted and tested as shown.

    ______________________________________                                        EXAMPLE 56                                                                    Appearance of Reconstituted Formulations                                      Formula-            Formula-                                                  tion A Appearance   tion B   Appearance                                       ______________________________________                                         4° C.                                                                        clear, blue-green                                                                           4° C.                                                                          clear, blue-green                                22° C.                                                                        clear, blue-green                                                                          22° C.                                                                          clear, blue-green                                30° C.                                                                        clear, blue-green                                                                          30° C.                                                                          clear, blue-green                                50° C.                                                                        clear, blue-green                                                                          50° C.                                                                          turbid, yellowish-brown                          ______________________________________                                    

HMW Determination Using SEHPLC

SEHPLC is performed by equilibrating a Shodex WS-803F column (0.8×30cm)in 0.1M Phosphate Buffer, pH 6.5, 0.15M Sodium Chloride at 1 mL/minflow rate and pumped through a detector set at 280 nm. This column iscapable of resolving differences in molecular weight based on itsfractionation range and exclusion limit (greater than 1 million MW).Under these conditions, the high molecular weight material elutes beforelow molecular weight material (salts and small molecular weightcomponents). The detector is connected to a computer that is programmedto integrate the peaks based on numerous parameters and quantify amountof the peaks based on a percent basis.

The % HMW species found and shown in Example 57 is a measure of theextent of polypeptide aggregation on storage. Example 57 also shows"Free Peg" (which is an indicator of the extent of hydrolysis of thesuccinate ester bond) and % enzyme activity.

    ______________________________________                                        EXAMPLE 57                                                                    Stability Studies with Lyophilized PEG-SOD                                    Formulations Containing 10% Sucrose or 10% HPβCD                         at 2 and 10 Months Stored at Various Temperatures.sup.1                       Formula-                                                                      tion   Tempera- HMW.sup.2  Free PEG.sup.3                                     (75,000                                                                              ture     (%)        (mg/ml)  % Activity.sup.4                          U/mg)  (°C.)                                                                           2 mo   10 mo 2 mo 10 mo 2 mo 10 mo                            ______________________________________                                        10%     4       0.3    0.2   0.35 0.1   99   --                               Sucrose                                                                              22       0.4    0.6   0.36 0     100  --                                      30       0.7    0.9   0.44 0     98   100                                     50       20     >50   3.45 ˜10                                                                           71   --                               10%     4       0.3    --    0.12 --    100  --                               HPβCD                                                                           22       0.4    --    0.08 --    99   --                                      30       0.4    0.7   0.14 0.9   100  100                                     50       0.6    4.8   0.87  3.23 95    92                              ______________________________________                                    

As can be seen from the data in Example 57, the HPβCD formulationdemonstrates excellent stability on storage at 30° C. No significantchange in enzyme activity was seen even after 10 months of storageindicating that satisfactory stability of the enzyme is maintained. Themarked superiority of HPβCD as a stability is evident by comparison ofthe % HMW values between the sucrose and the HPβCD formulations on 50°C. storage: the % HMW value for the HPβCD formulation after heatstressing at 50° C. for 10 months was only 4.8 as compared to >50 forthe sucrose formulation under identical conditions. The dramaticstabilizing ability of HPβCD even under high temperature storage isindicative of a unique stabilizing mechanism afforded by this stabilizerthat minimizes protein interaction and thus decreases the formation ofHMW species. It may be theorized that since cyclodextrin structurallycontains cavities, it is probable that the protective mechanism isrelated to encapsulation of certain reactive centers (associated withthe formation of HMW aggregates) which minimizes interaction andminimizes aggregation.

Result of stability studies with lyophilized PEG-catalase formulationscontaining 10% HPβCD were similar to that shown in Example 57.

The enhanced stability afforded by the present invention permits storageof the product at room temperatures and increases its shelf-life. Thislyophilized product is suitable for packaging in either a conventionalglass vial or in a prefilled syringe. The prefilled syringe offers a"ready to use" product that requires minimal handling and is well suitedfor emergency use. The formulations have great utility heretofore notprovided, in clinical, hospital and emergency situations.

The physician will determine the dosage of the present therapeuticformulations which will be most suitable and it will vary with theparticular formulation chosen, and furthermore, it will vary with theparticular patient under treatment. He will generally wish to initiatetreatment with small dosages substantially less than the optimum dose ofthe formulation and increase the dosage by small increments until theoptimum effect under the circumstances is reached. The formulations areuseful in the same manner as comparable therapeutic formulations and thedosage level is of the same order of magnitude as is generally employedwith these other therapeutic formulations. The therapeutic dosage forPEG-catalase when administered parenterally will generally be from 2,000to 20,000 IU/kg of body weight (International Units per kg), andpreferably from 5,000 to 10,000 IU/kg of body weight.

While preferred embodiments of the invention have been described andillustrated in the specification, it is to be understood that such ismerely illustrative of the underlying concept and features of theinvention and are not to be limiting of the scope of the invention andthe appended claims.

What is claimed is:
 1. A reconstituted lyophilized formulation ofpolyethylene glycol and a biologically active catalase conjugatecomprising:a) polyethylene glycol having a molecular weight of fromabout 1,000 to about 15,000 daltons and consisting of less than about10% w/w non-monomethoxylated polyethylene glycol and more than 90% w/wmonomethoxylated polyethylene glycol; said conjugate having an enzymaticactivity of from about 150 to about 150,000 U/ml; b) from about 0.1 toabout 20% w/v of cyclodextrin; and c) from about 0.01 to about 50 nMbuffer, said formulation having a pH of from about 5.7 to about 6.5. 2.The reconstituted lyophilized formulation of claim 1 wherein saidconjugate has an enzymatic activity of from about 25,000 to about150,000 U/ml.
 3. The reconstituted lyophilized formulation of claim 1wherein said conjugate has an enzymatic activity of from about 50,000 toabout 150,000 U/ml.
 4. The reconstituted lyophilized formulation ofclaim 1 wherein said cyclodextrin is present in an amount of from about1.0 to about 15% w/v.
 5. The reconstituted lyophilized formulation ofclaim 1 wherein said cyclodextrin is present in an amount of from about5 to about 10% w/v.
 6. The lyophilized formulation of claim 1 whereinsaid polyethylene glycol has an average molecular weight of from about2,000 to about 10,000 daltons.
 7. The lyophilized formulation of claim 1wherein said polyethylene glycol has an average molecular weight of fromabout 4,000 to about 6,000 daltons.
 8. The lyophilized formulation ofclaim 1 wherein said polyethylene glycol contains less than about 7% w/wnon-monomethoxylated polyethylene glycol.
 9. The lyophilized formulationof claim 1 wherein said polyethylene glycol contains less than about 5%w/w non-monomethoxylated polyethylene glycol.
 10. The lyophilizedformulation of claim 1 wherein said cyclodextrin is a derivative ofβ-cyclodextrin.
 11. The lyophilized formulation of claim 10 wherein saidβ-cyclodextrin derivative is hydroxypropyl cyclodextrin.
 12. Thelyophilized formulation of claim 10 wherein said β-cyclodextrinderivative is glucosyl cyclodextrin.
 13. The lyophilized formulation ofclaim 10 wherein said β-cyclodextrin derivative is maltosylcyclodextrin.
 14. The lyophilized formulation of claim 10 wherein saidβ-cyclodextrin derivative is maltotriosyl cyclodextrin.
 15. Thelyophilized formulation of claim 1 wherein said catalase is bovinehepatocatalase.
 16. A process of preparing a lyophilized biologicallyactive catalase composition comprising the steps of:a) carboxylatingpolyethylene glycol containing less than 10% w/w non-monomethoxylatedpolyethylene glycol; b) activating said carboxylated polyethylene glycolto obtain an active polyethylene glycol ester; c) covalently attachingsaid active polyethylene glycol ester to a biologically active catalase;d) solubilizing said covalently attached polyethylene glycol ester andsaid biologically active catalase in an aqueous media; e) solubilizingcyclodextrin in said aqueous media to obtain a homogeneous solution; f)buffering said solution to a pH of from about 5.7 to about 6.5; and h)lyophilizing the solution.
 17. The process of claim 16 wherein saidsolution for lyophilization comprises:from about 150 to about 150,000unit/ml of a covalently bound low diol polyethylene glycol/catalase;from about 0.1 to about 20% w/v of cyclodextrin; and from about 0.01 toabout 50 mM of a buffer.
 18. The process of claim 16 wherein saidpolyethylene glycol has an average molecular weight of from about 1,000to about 15,000 daltons.
 19. The process of claim 16 wherein saidcyclodextrin is a derivative of β-cyclodextrin.
 20. The process of claim19 wherein said β-cyclodextrin derivative is hydroxypropyl cyclodextrin.21. The process of claim 19 wherein said β-cyclodextrin derivative ismaltosyl cyclodextrin.
 22. The process of claim 19 wherein saidβ-cyclodextrin derivative is maltotriosyl cyclodextrin.
 23. A method oftreating a disease condition caused by hydrogen peroxide anions ontissue in a mammal comprising administering an effective amount of acomposition according to claim
 1. 24. The method of claim 23 whereinsaid disease condition is inflammation.
 25. The method of claim 23wherein said disease condition is ischemia.
 26. The method of claim 23wherein said disease condition is reperfusion injury.
 27. The method ofclaim 23 wherein said disease condition is trauma.
 28. The method ofclaim 23 wherein said disease condition is stroke.